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Abstract

Multiphoton excitation by temporally focused pulses can be combined with spatial Fourier-transform pulse shaping techniques to enhance spatial control of the excitation volume. Here we propose and demonstrate an optical system for the generation of such spatiotemporally engineered light pulses using a combination of spatial control by a two-dimensional reconfigurable light modulator, with a dispersive optical setup for temporal focusing. We show that although the properties of a holographic beam significantly differ from those of plane-wave illumination used in previous temporal focusing realizations, this leads only to a slightly reduced axial resolution. We show that the system can provide scanningless, arbitrarily shaped, depth resolved excitation patterns that offer new perspectives for multiphoton photoactivation and optical lithography applications.

Figures (3)

(a) Layout of the experimental setup. (b) Images of 10, 20 and 30µm diameter spots, obtained by two-photon excitation of a ~0.9µm thick spin-coated fluorescent layer. (c) A confocal image is recorded from a Purkinje cell loaded by Alexa 535. A selected region of interest (dashed box) is extracted and sent as the input for the calculation algorithm. (d) Phase-hologram generated by the algorithm (e) Illumination spot generated at the objective focal plane, with the phase-hologram of (d), visualized by exciting the thin fluorescent layer. (f) Overlay of (c) and (e).

(a) Images (100ms integration time) of a holographic shape visualized by exciting a thin fluorescent layer without (left) and with (right) a rotating diffuser positioned after the dispersion grating. 40 different locations of the diffuser were averaged in the 100 ms of the exposure time. (b) Lateral intensity profile of the excited shape along the yellow lines of Fig. 3(a) right (black line) and left (red line). The intensity variation around a mean value A is ~50% for the black line and is reduced to 10% with the rotating diffuser.